Compositions comprising purified 2-methoxyestradiol and methods of producing same

ABSTRACT

2-methoxyestradiol having greater than 98% purity is obtained by synthetic or purification methods. This highly pure 2-methoxy estradiol, lacking estrogenic components, is particularly suitable for clinical use in humans. The purification methods of the invention involve the use of liquid-solid chromatography (LSC) to separate 2-ME2 from other compounds. The chromatographic media is preferably silica. The solvent system comprises a non-polar solvent, such as chloroform, and a polar solvent, such as methanol.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/150,293 filed Aug. 23, 1999.

FIELD OF THE INVENTION

The invention relates to the estradiol metabolite 2-methoxyestradiol andto methods of obtaining purified 2-methoxyestradiol.

BACKGROUND OF THE INVENTION

2-Methoxyestradiol, 1,3,5(10)-estratrien-2,3,17β-triol-2-methyl-ether(2-ME2) is an endogenous metabolite of estradiol, the major ovarianestrogen. The chemical formula of 2-ME2 is C₁₉H₂₆O₃, and the compoundhas a molecular weight of 302.4. 2-ME2 has low of estrogenic activitybut has been found to have other biological effects.

U.S. Pat. Nos. 5,504,074, 5,661,143, and 5,892,069 to D'Amato et al.disclose methods of treating mammalian diseases characterized byabnormal cell mitosis using 2-ME2. Undesirable cell mitosis ischaracteristic of many diseases, including, but not limited to, cancer,atherosclerosis, proliferation of solid tumors, vascular malfunctions,endometriosis, retinopathies, arthropathies, and abnormal wound healing.In addition, cell mitosis is important in a wide variety of biologicalfunctions, including but not limited to the normal development of theembryo, formation of the corpus luteum, cyclic proliferation of uterineendometrium, wound healing, and inflammatory and immune responses.

U.S. Pat. No. 5,521,168 to Clark discloses using 2-ME2 for loweringintraocular pressure. 2-ME2 also inhibits estrogen-induced pituitarytumor angiogenesis and suppresses tumor growth in Fisher 344 rats asreported by Banerjee, S. K. et al., Proc. Amer. Assoc. Cancer Res. 39,March 1998.

Presently, commercially available preparations of 2-ME2 are either lessthan 98% pure or contain undesirable steroid contaminants that are ofconcern for pharmaceutical uses. Important contaminants of thesepreparations are estradiol, 4-hydroxyestradiol, 4-methoxyestradiol,2-hydroxyestradiol, estrone, and 2-methoxyestrone. The amounts of thesecontaminants that are found in presently available 2-ME2 preparationsare unacceptable for pharmaceutical applications.

Any therapeutic use of 2-ME2 in humans requires 2-ME2 having a highlevel of purity. In general, therapeutic agents are required to besubstantially pure to avoid negative side effects of contaminants. Inparticular, since 2-ME2 has effects that are counteracted by estradioland other estrogenic metabolites, it is crucial to have a 2-ME2preparation substantially free of such contaminants. Effects that may beseen from contaminating estradiol, estrone, and 2-hydroxyestradiolinclude estrogenic effects such as feminization, endometrialproliferation, increased risk of uterine and breast cancer,developmental effects on sexual organs, inhibition of leukopoesis, andeffects on hematopoetic cells. 4-hydroxyestradiol, 4-methoxyestradiol,and estradiol are known mutagens and carcinogens.

Accordingly, what is needed is a composition of 2-ME2 which is greaterthan 98% pure and which contains substantially no estradiol or othersteroids having estrogenic or carcinogenic effects.

What is also needed is a composition containing 2-ME2 that is greaterthan 99.5% pure.

What is also needed are methods for making 2-ME2 of greater than 98%purity and containing substantially no estradiol or other steroidshaving estrogenic or carcinogenic effects.

Also needed are methods of substantially separating 2-ME2 fromestradiol, related molecules, and other contaminants, resulting in 2-ME2having a purity of greater than 99.5%.

SUMMARY OF THE INVENTION

The present invention provides 2-ME2 having greater than 98% purity,more preferably greater than 99% purity, most preferably greater than99.5% purity. The 2-ME2 preparations preferably contain less than 0.03%estradiol, 0.02% or less 2-hydroxyestradiol, 0.02% or less4-hydroxyestradiol, 0.02% or less 4-methoxyestradiol, and less than0.02% estrone. More preferably, the 2-ME2 preparations contain 0.01% orless estradiol, 0.02% or less 2-hydroxyestradiol, 0.01% or less4-hydroxyestradiol, 0.01% or less 4-methoxyestradiol, and 0.01% or lessestrone.

The present invention also provides methods of obtaining 2-ME2 ofgreater than 98% purity, more preferably greater than 99% purity, mostpreferably greater than 99.5% purity. In some embodiments, the methodsinvolve synthetic techniques. In other embodiments, the methods involvepurification techniques to separate the 2-ME2 from other compounds. Inyet other embodiments, the methods involve both synthetic techniques andpurification techniques described herein.

The purification methods involve the use of liquid-solid chromatography(LSC) to separate 2-ME2 from other compounds. The chromatographic mediais preferably silica. The solvent system comprises a non-polar solvent,such as chloroform, and a polar solvent, such as methanol.

Accordingly, an object of the present invention is to provide 2-ME2having a purity greater than 98%.

Another object of the present invention is to provide 2-ME2substantially free of estradiol, related compounds, and other unwantedimpurities.

Still another object of the invention is to provide methods of obtainingsubstantially pure 2-ME2 by synthetic techniques.

Another object of the invention is to provide methods of obtainingsubstantially pure 2-ME2 by purification techniques.

Other features and advantages of the invention will be apparent from thefollowing description of preferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chromatogram from the reversed phase HPLC analysis of2-methoxyestradiol available from Sigma Chemical Company (45H4033). Thisgraph shows that the Sigma product contains about 0.034% estradiol.

FIG. 2 is an expanded view of the chromatogram in FIG. 1 indicating theestradiol impurity.

FIG. 3 is a chromatogram from the reversed phase HPLC analysis of2-methoxyestradiol available from Research Plus (10699). This graphshows that the Research Plus product contains about 0.024% estrone andabout 0.93% other undesirable estrogens.

FIG. 4 is an expanded view of the chromatogram in FIG. 3 indicating theestrone impurity.

FIG. 5 is a chromatogram from the reversed phase HPLC analysis of theunpurified 2-methoxyestradiol employed as the starting material inExample 2 of the present invention.

FIG. 6 is a chromatogram of the 2-ME2 of the present invention producedin Example 2. The HPLC was run with a non-overloaded amount of sample,75.6 μg (14 μl at 5.4 μl/ml).

FIG. 7 is an expanded view of the chromatogram in FIG. 6.

FIG. 8 is a chromatogram of the 2-ME2 of the present invention producedin Example 2. The HPLC was run with an overloaded amount of sample, 270μg (50 μl at 5.4 μl/ml).

FIG. 9 is an expanded view of the chromatogram in FIG. 8.

FIG. 10 is a chromatogram of the pooled fractions from Example 2,assayed using a gradient (20 to 70% acetonitrile over 25 minutes, 1%acetic acid, and remainder water). 43.2 μg (8 μl of the 5.4 μl/mlsample) was injected.

FIG. 11 is an expanded view of the chromatogram in FIG. 10.

FIG. 12 depicts a synthetic reaction scheme for the production of the2-methoxyestradiol of the present invention, using estradiol as astarting material and employing bromine, a crown ether, and a blockinggroup on the 3- and 17-position hydroxyloxygen atoms of the estradiol.

FIG. 13 depicts a synthetic reaction scheme for the production of the2-methoxyestradiol of the present invention, using estradiol as astarting material and employing bromination at the 2-position of the Aring of unblocked estradiol and a crown ether.

FIG. 14 depicts a synthetic reaction scheme for the production of the2-methoxyestradiol of the present invention, using estradiol as astarting material and employing a blocking group on the 3- and17-position hydroxyloxygen atoms of estradiol, nitration, and aSandmeyer reaction.

FIG. 15 depicts a synthetic reaction scheme for the production of the2-methoxyestradiol of the present invention, using estrone as a startingmaterial and employing a blocking group on the 3-position hydroxyloxygenatom, nitration, and a Sandmeyer reaction.

FIG. 16 depicts a synthetic reaction scheme for the production of the2-methoxyestradiol of the present invention, using estradiol as astarting material and employing bromination at the 2-position of the Aring of unblocked estradiol and reaction with methanol.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to 2-methoxyestradiol having a purityof greater than 98.0%, more preferably greater than 99.0%, and mostpreferably of 99.5% or higher. 2-ME2 can be obtained through synthesismethods or purification methods described herein that yield highly pure2-ME2. The synthesis methods described herein may also be supplementedwith the purification methods described herein to yield 2-ME2 havingeven greater purity.

Although the terms “2-methoxyestradiol” and 2-ME2 are specifically usedherein, it should be understood that the methods disclosed herein can beused for synthesis or purification of other compounds, such as, but notlimited to, estradiol and other structurally related steroids.

Methods of Synthesis

The present invention provides methods of synthesizing 2-ME2 to a purityof greater than 98.0%, more preferably greater than 99.0%, and mostpreferably of 99.5% or higher. The synthetic methods described hereincan also be used, with minor modifications, to synthesize other 2- and4-derivatives or analogues of estradiol, such as, for example,4-methoxyestradiol and 4-hydroxyestradiol.

There are several synthetic approaches that can be taken to prepare2-ME2 having a purity greater than 98.0%. Alternatively, 2-ME2 can bepurified according to the following purification methods to have apurity greater than 98.0%. These different synthetic approaches utilizedifferent starting materials and intermediates; consequently, differentyields, side reactions and impurities will be obtained.

Two similar approaches employ estradiol as a starting material andutilize a brominated intermediate, as taught by Rao, P. N. et al.,Synthesis, 1977, 168 and Chen, S-H et al., Steroids, 1986, 47, 63. Thefirst approach is illustrated in FIG. 12. The free hydroxyl groups ofestradiol are protected with a blocking group. A wide range of blockinggroups can be used in the present invention. These blocking groupsinclude, but are not limited to, alkyl, aryl, aralkyl groups, and alkyl,aryl, and aralkyl group containing one or more heteroatoms. For example,protection can be accomplished using an alkyl halide, such as benzylbromide, to form an alkyl ether. Appropriate conditions for hydroxylprotection include reaction of the estradiol and alkyl halide in thepresence of NaH and TBAI, optionally in the presence of a solvent, suchas dimethyl formamide (DMF). The protected estradiol is then reactedwith bromine, for example, in the presence of acetic acid. Protection ofthe free hydroxyls during bromination gives a higher yield of the2-brominated intermediate (about 70% vs. about 20% without theprotecting groups) (see Cushman, M. et al., J. Med. Chem. 1997, 40,2323).

The bromine is then replaced with a methoxide group using a coppercatalyst. For example, the brominated intermediate can be reacted withNaOMe in the presence of a copper catalyst, such as CuI. The reaction ispreferably conducted in a solvent, such as DMF, optionally in thepresence of a promoter. Acceptable promoters, include, but are notlimited to, crown ethers, such as benzo-15-crown-5.

Removal of the protecting groups, for example, by catalytichydrogenation of the alkyl moiety, yields 2-ME2. Unfortunately, thissynthetic route yields about 1–2% impurity of estradiol from themethoxylation step (a hydride quenches the reactive copper complexrather than a methoxide). The estradiol can be removed to undetectablelevels by chromatography, such as described below, or significantlyreduced by successive crystallization in chloroform.

Another synthetic method utilizing a brominated intermediate andemploying estradiol as the starting material is illustrated by FIG. 13.In this synthetic reaction, the estradiol is ring brominated withoutfirst blocking the hydroxyl groups. The bromine is then replaced with amethoxide using a copper catalyst in a manner similar to that describedabove.

In another approach, estradiol or estrone can be used as a startingmaterial in a reaction scheme that utilizes nitro/amine intermediates(see Cushman, M. et al., J. Med. Chem. 1995, 38, 2041). These syntheticapproaches are illustrated in FIG. 14 (estradiol starting material) andFIG. 15 (estrone starting material). In these approaches, the freehydroxyl groups are protected. This protection can be accomplished, forexample, using an alkyl halide, such as benzyl bromide, to form an alkylether. Appropriate conditions for hydroxyl protection include reactionof the starting material and alkyl halide in the presence of NaH andTBAI, optionally in the presence of a solvent, such as dimethylformamide (DMF).

The protected starting material is then nitrated, for example, withnitric acid and acetic acid or with nitric acid and sulfuric acid, toform the corresponding 2-nitro product. The nitro group is then reduced.Selective reduction can be accomplished by catalytic hydrogenation, forexample, hydrogenation in the presence of Pd/C to produce thecorresponding 2-amine. The catalytic reduction is optimally carried outfor a period of one hour. Using Sandmeyer conditions (nitrous acid andsodium methoxide), the 2-amino group can be converted to the 2-methoxysubstituent. Catalytic hydrogenation removes the protecting groups togive 2-ME2 when the starting material is estradiol and 2-methoxyestronewhen the starting material is estrone. Reduction of the 17-keto group of2-methoxyestrone with sodium borohydride yields 2-ME2.

Yet another method employs estradiol as the starting material andutilizes brominated intermediates. In this synthetic reaction, theestradiol is ring brominated without first blocking the hydroxyl groups.Bromination is accomplished, for example, with bromine and acetic acidin a solvent, such as THF. This reaction results in bromination atdifferent sites on the ring, including multi-brominated species. The2-bromo-estradiol can then be isolated from the other brominatedintermediates, for example, by chromotography or crystallization,followed by replacement of the bromine with a methoxide. The bromine canbe replaced with a methoxide group, for example, using sodium methoxideand methanol in the presence of a copper catalyst, such as CuI, in amanner similar to that described above. Alternatively, the intermediatescan be reacted to form the corresponding methoxides, followed byisolation of the 2-methoxyestradiol by the methods described above.

Methods of Purification

The present invention provides methods of purifying 2-ME2 to a purity ofgreater than 98.0%, more preferably greater than 99.0% and mostpreferably of 99.5% or higher. The 2-ME2 preparations preferably containless than 0.03% estradiol, 0.02% or less 2-hydroxyestradiol, 0.02% orless 4-hydroxyestradiol, 0.02% or less 4-methoxyestradiol, and less than0.02% estrone. Most preferably, the 2-ME2 preparations contain 0.01% orless estradiol, 0.02% or less 2-hydroxyestradiol, 0.01% or less4-hydroxyestradiol, 0.01% or less 4-methoxyestradiol, and 0.01% or lessestrone.

The purification methods of the present invention involve liquidchromatography on an adsorption/partition medium such as silica, using asolvent system comprising a polar and a non-polar solvent. Thepurification methods described herein can also be used, with minormodifications, to purify compounds similar to 2-ME2, such as, forexample, 4-methoxyestradiol, 4-hydroxyestradiol, 2-hydroxyestradiol,estradiol, estrone, 2-methoxyestrone, and 4-methoxyestrone.

The Sample

The sample to be purified can be synthesized, or obtained from abiological source. The sample may be a commercially available 2-ME2preparation, such as those sold by Sigma-Aldrich Chemicals of St. Louis,Mo., Research Plus, Inc. of Bayonne, N.J., or Calbiochem of San Diego,Calif. The sample is preferably at least about 50% pure, more preferablyabout 75% pure, even more preferably about 90% pure, and most preferablyabout 98% pure. The sample can be subjected to other purification stepsprior to the methods described herein, such as selectivecrystallization.

The sample is preferably dissolved into or solvent-exchanged into aloading solvent, as further described below. The sample is preferably ata concentration in the range of about 0.01 to 2 g/ml, preferably about0.01 to 1 g/ml, more preferably about 0.05 to 0.2 g/ml.

Chromatographic Media

Silica is preferably used as the chromatographic medium. Silica gel ofabout 70–400 mesh is preferred, most preferably about 70–230 mesh, suchas supplied by Merck and other vendors. The medium can be used loose, inbatch chromatography, or packed into a column. Pre-packed columns, suchas those sold by Biotage of Charlottesville, Va., can also be used. Themedium should be equilibrated in an appropriate solvent beforeapplication of the sample to the medium, as further discussed below.

Column Dimensions

The chromatographic methods described herein can be achieved using batchor column chromatography. In batch chromatography, the sample and thechromatographic medium are combined in a container for a period of timesufficient to allow the 2-ME2 to be retained by the medium. The mediumis then preferably washed with wash solvent. Elution solvents are thenapplied to the medium. After the loading, wash, and elution steps, thesolvent is removed from the medium, such as by filtration.

For column chromatography, a column having appropriate dimensions ispacked with the chromatography medium. The column, after equilibrationwith appropriate solvent, is loaded with sample by applying the sampleto the top, or entrance, of the column. The ratio of the sample volumeto column diameter should preferably be between about 0.2 to 3 ml/cm,and more preferably between about 0.5 and 1.5 ml/cm for best results.

Solvents

A solvent system including a polar solvent, such as methanol (MeOH), anda non-polar solvent, such as chloroform (CHCl₃), is used. Other polarsolvents that can be used include, but are not limited to,tetrahydrofuran (THF), ethyl acetate, isopropanol, ethanol, propanol,and combinations thereof. Other non-polar solvents that can be usedinclude, but are not limited to, hexane, dichloromethane, cyclohexane,pentane, and combinations thereof. More specifically, solvent systemsthat can be used include THF/hexane, ethyl acetate/hexane,isopropanol/hexane, ethanol/CHCl₃, propanol/CHCl₃, isopropanol/CHCl₃,and combinations thereof.

The sample is soluble in the polar solvent. Some amount of the polarsolvent, generally about 10%, is needed to render the sample soluble inthe loading solvent. The loading solvent thus will include up to about10% polar solvent and about 90% non-polar solvent.

After the sample is loaded onto the medium, the medium may be washedwith a wash solvent that will wash contaminants off the medium but willnot elute the 2-ME2. The wash solvent comprises mostly non-polarsolvent, with enough polar solvent to prevent the 2-ME2 fromprecipitating but not enough polar solvent to elute the 2-ME2.

The sample is eluted with elution solvent that contains enough polarsolvent to elute the 2-ME2 from the medium. The elution solvent may beapplied as a step gradient or as a linear gradient, as described below.

Column Conditions

The wash and elution solvents can be applied to the column in a stepgradient or in a linear gradient. In a preferred embodiment, thesolvents are applied using a step gradient of increasing concentrationof polar solvent.

The column can be operated using the force of gravity or can be operatedwith a pump that forces liquids through the column. The rate at whichthe column is operated will depend upon the volume and dimensions of thecolumn and the silica gel particle size. In general, the column can beoperated at a rate from about 0.5 to 5 ml/min.

The eluant can be monitored visually or, monitored with aspectrophotometer at a wavelength of about 288 nm, which is theabsorbance maximum of 2-ME2, and collected as the 2-ME2 elutes from thecolumn.

The column can optionally be operated under pressure and can optionallybe heated. Preparative high performance liquid chromatography (HPLC),either normal phase or reversed phase, or fast performance liquidchromatography (FPLC) techniques can be used. Commercial preparativechromatography apparatus, such as that sold by Biotage ofCharlottesville, Va., can also be used. Other known methods of improvingcolumn efficiency and/or speed can also be employed.

Sample Collection and Treatment

The eluant can be collected as fractions which are then assayed for2-ME2 content and purity. These fractions can then be combined toachieve the desired purity of 2-ME2. The fractions can be assayed forpurity using reverse phase HPLC with a C-18 column (Waters) and anisocratic solvent system of 30:69:1 acetonitrile:water:acetic acid.Other systems can be used for sample analysis, such those that usesolvent gradients instead of the isocratic solvent system; those thatuse trifluoroacetic acid or formic acid rather than acetic acid; andthose that use methanol rather than acetonitrile.

Alternatively, or in combination, the eluant can be monitored in realtime and sample collection begun when 2-ME2 of desired purity elutesfrom the column.

The solvent is removed from the pooled fractions by use of a vacuumand/or other solvent removal methods. Lyophilization and otherevaporative methods can be used.

PREFERRED EMBODIMENT

In a preferred embodiment the medium is silica, which is packed into acolumn. The sample is dissolved in a mixture of CHCl₃ and MeOH, withenough MeOH to solubilize the 2-ME2, generally about 90:10 CHCl₃:MeOH.The elution conditions are a step gradient from 99:1 CHCl₃:MeOH to 98:2CHCl₃:MeOH.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLE 1

Commercially available samples of 2-ME2 were assayed by analytical HPLCto determine their overall purity and the amounts of certaincontaminants, namely estradiol, 4-hydroxyestradiol, 4-methoxyestradiol,2-hydroxyestradiol, estrone, and 2-methoxyestrone.

These analytical HPLC chromatograms were generated using reverse phaseHPLC with a C-18 column (Waters) and a solvent gradient (20 to 50%acetonitrile over 30 minutes, 50 to 80% acetonitrile over 5 minutes, 1%acetic acid, remainder water). The eluant was monitored at a wavelengthof 288 nm. In this system 2-ME2 elutes at about 21.5 minutes, estradiolelutes at about 20.0 minutes, estrone elutes at about 23.2 minutes,4-hydroxyestradiol elutes at about 15.0 minutes, 4-methoxyestradiolelutes at about 20.4 minutes, 2-hydroxyestradiol elutes at about 15.4minutes, and 2-methoxyestrone elutes at about 24.4 minutes.

The chromatogram of a sample from Sigma-Aldrich Chemicals of St. Louis,Mo. is shown in FIG. 1. The sample has an overall purity of 99.2% buthas contaminating estradiol of about 0.034%, an unacceptable amount.FIG. 2 is an expanded view of the chromatogram of FIG. 1.

FIG. 3 is a chromatogram of a sample obtained from Research Plus, Inc.of Bayonne, N.J. that shows that the 2-ME2 has a purity of 98.6%. Theautomatic peak calculator and the expanded view shown in FIG. 4 showthat the preparation contains 0.024% estrone, an unacceptable amount ofthis contaminant. Other samples tested showed 2-ME2 purity less than98%, including a second batch obtained from Research Plus (97.2% 2-ME2)and a sample from CalBiochem of San Diego, Calif. (91.8% 2-ME2).

Table 1, below, illustrates the purity and contaminants of thesecommercially available samples of 2-ME2 and the purified 2-ME2 of thepresent invention.

TABLE 1 Re- Research search Plus, Plus, Calbio- Pharm- puri- Sigma Lot#1 Lot #2 chem Eco fied 2-ME2 99.18  98.61 97.17  91.80  97.80 99.98estradiol 0.03 n.d. n.d. 1.78 2.2 less than 0.01% estrone n.d.  0.020.43  0.011 n.d. 4-hydroxy- n.d. n.d. n.d. n.d. n.d. estradiol4-methoxy- 0.49  0.121 0.18 1.99 n.d. estradiol 2-hydroxy- n.d. n.d.n.d. 0.06 n.d. estradiol 2-methoxy- n.d. n.d. n.d. 0.20 n.d. estrone*n.d. means none was detected.

EXAMPLE 2

A 55 cm diameter (60 cm height) glass column was packed with 600 gsilica gel (70–230 mesh from Merck) in 90:10 CHCl₃:MeOH. The column waswashed with one liter of CHCl₃ to remove the MeOH from the column.

The sample was 3.5 g 2-ME2 in 60 ml 90:10 CHCl₃:MeOH. The 2-ME2 wasobtained from PharmEco Laboratories, Inc. of Lexington, Mass., and was97.8% pure as determined by analytical HPLC (FIG. 5). The peak elutingat 10.917 is estradiol (2.2%).

Analytical HPLC of the starting material, the column fractions, and thepooled product was performed using reverse phase HPLC with a C-18 column(Waters) and an isocratic gradient of 30:69:1 acetonitrile:water:aceticacid, which provides good separation of 2-ME2 and estradiol. The eluantwas monitored at a wavelength of 288 nm.

The sample was applied to the top of the column and allowed to enter thebed volume. The column was eluted with one liter of 99:1 CHCl₃:MeOH andthen 1.5 L of 98:2 CHCl₃:MeOH. Fractions of 50 ml each were collectedand 15 fractions containing 2-ME2 were assayed for 2-ME2 purity usingthe analytical isocratic HPLC system described above. Nine to tenfractions that showed no amount of estradiol were pooled together andsolvent was evaporated. After drying under vacuum for 4 hours, 3.2 g ofyellow/white crystals were collected, for a 91% yield.

Purity of the pooled fractions was determined by analytical HPLC to be99.984%, using the isocratic technique described above. The HPLCchromatograms are shown in FIGS. 6 through 9. FIG. 6 was generated witha non-overloaded amount of sample, 75.6 μg (14 μl at 5.4 μl/ml). FIG. 7is an expanded view of the chromatogram of FIG. 6. The automatic peakfinder calculated the 2-ME2 to be 100.0%, although a small, unknownimpurity peak is seen in the expanded view, eluted prior to the 2-ME2.FIG. 8 was generated with an overloaded amount of sample, 270 μg (50 μlat 5.4 μl/ml). FIG. 9 is an expanded view of the chromatogram of FIG. 8.The automatic peak finder calculated the 2-ME2 to be 99.984% pure, witha small, unknown, impurity that eluted prior to the 2-ME2, and afterestradiol, that was calculated to be 0.016%. The expanded view shown inFIG. 9 shows this impurity peak more clearly and shows that the 2-ME2peak is very clean.

The pool was also assayed using a gradient (20 to 70% acetonitrile over25 minutes, 1% acetic acid, and remainder water). 43.2 μl (8 μl of the5.4 μl/ml sample) was injected. The chromatogram is shown in FIG. 10.The automatic peak finder calculated the 2-ME2 to have a purity of99.825%. However, when an artifact peak, present in a blank run, at29.45 minutes is removed from consideration, the calculated purity is99.9%. FIG. 11 is an expanded view of this chromatogram. The unknownimpurity at 13.43 minutes was calculated to be 0.012%. If estradiol werepresent, it would elute between the unknown purity and the 2-ME2 peak.If estradiol is present, therefore, it can be present at no more than ⅓to ¼ of the amount of the unknown peak. Accordingly, the estradiolamount was estimated to be no more than 0.004%. The preparationcontained 0.02% or less 2-hydroxy-estradiol, 0.01% or less4-hydroxy-estradiol, 0.01% or less 4-methoxy-estradiol, and 0.01% orless estrone, as demonstrated by the lack of any measurable peaks at theexpected retention times.

The purified sample was also subjected to elemental analysis and theresults are shown in Table 2.

TABLE 2 Elemental Analysis Element Theoretical Found Carbon 75.46 75.21Hydrogen 8.67  8.65 Oxygen 15.87 16.13 (obtained by difference) Chlorine0.00 0.0

The above description is intended to be illustrative and notrestrictive. Many embodiments will be apparent to those of skill in theart upon reading the above description. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to theappended claims, along with the full scope of equivalents to which suchclaims are entitled. The disclosures of all articles and references,including patents, patent applications and publications, areincorporated herein by reference.

1. A pharmaceutical composition being substantially free of steroidcontaminants having estrogenic or carcinogenic effects comprising2-methoxyestradiol having a purity greater than 99.5% as determined byHPLC.
 2. The composition of claim 1, containing less than 0.03%estradiol and less than 0.02% estrone.
 3. The composition of claim 2,containing less than 0.01% estradiol and less than 0.01% estrone.
 4. Thecomposition of claim 2, further containing less than 0.02%2-hydroxyestradiol.
 5. The composition of claim 2, further containingless than 0.02% 4-hydroxyestradiol.
 6. The composition of claim 2,further containing less than 0.02% 4-methoxyestradiol.
 7. Thecomposition of claim 1, containing 0.01% or less estradiol, 0.02% orless 2-hydroxyestradiol, 0.01% or less 4-hydroxyestradiol, 0.01% or less4-methoxyestradiol and 0.01% or less estrone.
 8. A pharmaceuticalcomposition being substantially free of steroid contaminants havingestrogenic or carcinogenic effects comprising 2-methoxyestradiol havinga purity greater than 98.0% and containing less than 0.03% estradiol andless than 0.02% estrone.
 9. The composition of claim 8, containing lessthan 0.01% estradiol and less than 0.01% estrone.
 10. The composition ofclaim 8, containing 0.01% or less estradiol, 0.02% or less2-hydroxyestradiol, 0.01% or less 4-hydroxyestradiol, 0.01% or less4-methoxyestradiol and 0.01% or less estrone.
 11. The composition ofclaim 8, wherein the 2-methoxyestradiol has a purity greater than 99.0%.12. The composition of claim 11, containing less than 0.01% estradioland less than 0.01% estrone.
 13. The composition of claim 11, containing0.01% or less estradiol, 0.02% or less 2-hydroxyestradiol, 0.01% or less4-hydroxyestradiol, 0.01% or less 4-methoxyestradiol and 0.01% or lessestrone.
 14. A method for purifying 2-methoxyestradiol to a produce a2-methoxyestradiol substantially free of steroid contaminants havingestrogenic or carcinogenic effects and having a purity greater than 98%and containing less than 0.03% estradiol and less than 0.02% estronecomprising: adding a solution comprising 2-methoxyestradiol to achromatography medium; and eluting the 2-methoxyestradiol off of themedium with a solvent system comprising a polar solvent and a non-polarsolvent.
 15. The method of claim 14, wherein the medium is silica andwherein the 2-methoxyestradiol is eluted using a step gradient of 99:1CHCl₃:MeOH to 98:2 CHCl₃:MeOH.
 16. A method for producing2-methoxyestradiol substantially free of steroid contaminants havingestrogenic or carcinogenic effects and having a purity greater than 98%and containing less than 0.03% estradiol and less than 0.02% estronecomprising: protecting the 3- and 17-hydroxyl groups of estradiol;reacting the protected estradiol with bromine and acetic acid to producea 2-brominated derivative of estradiol; reacting the 2-brominatedderivative of estradiol with sodium methoxide in the presence of acopper catalyst; removing the protecting groups on the 3- and17-hydroxyl groups to produce 2-methoxyestradiol; and purifying the2-methoxyestradiol using liquid chromatography on anadsorption/partition medium with a solvent system comprising a polar anda nonpolar solvent.
 17. A method for producing 2-methoxyestradiolsubstantially free of steroid contaminants having estrogenic orcarcinogenic effects and having a purity greater than 98% and containingless than 0.03% estradiol and less than 0.02% estrone comprising:ring-brominating estradiol by reacting estradiol with bromine in thepresence of acetic acid to produce a ring-brominated intermediate;reacting the ring-brominated intermediate with sodium methoxide in thepresent of a copper catalyst to produce 2-methoxyestradiol; andpurifying the 2-methoxyestradiol using liquid chromatography on anadsorption/partition medium with a solvent system comprising a polar anda nonpolar solvent.
 18. A method for producing 2-methoxyestradiolsubstantially free of steroid contaminants having estrogenic orcarcinogenic effects and having a purity greater than 98% and containingless than 0.03% estradiol and less than 0.02% estrone comprising:protecting the 3- and 17-hydroxyl groups of estradiol; reacting theprotected estradiol with nitric acid and acetic acid to produce a2-nitro derivative of estradiol; reducing the 2-nitro derivative ofestradiol to produce the corresponding 2-amino derivative of estradiol;reacting the 2-amino derivative of estradiol under Sandmeyer conditionsto produce a 3-,17-hydroxyl protected 2-methoxyestradiol; and removingthe protecting groups on the 3- and 17-hydroxyl groups to produce2-methoxyestradiol.
 19. A method for producing 2-methoxyestradiolsubstantially free of steroid contaminants having estrogenic orcarcinogenic effects and having a purity greater than 98% and containingless than 0.03% estradiol and less than 0.02% estrone comprising:protecting the 3-hydroxyl group of estrone; reacting the protectedestrone with nitric acid and acetic acid to produce a 2-nitro derivativeof estrone; reducing the 2-nitro derivative of estrone to produce thecorresponding 2-amino derivative of estrone; reacting the 2-aminoderivative of estrone under Sandmeyer conditions to produce a 3-hydroxylprotected 2-methoxyestrone; removing the protecting group on the3-hydroxyl group to produce 2-methoxyestrone; and reducing the 17-ketogroup of 2-methoxyestrone to produce 2-methoxyestradiol.
 20. A methodfor producing 2-methoxyestradiol substantially free of steroidcontaminants having estrogenic or carcinogenic effects and having apurity greater than 98% and containing less than 0.03% estradiol andless than 0.02% estrone comprising: brominating estradiol in thepresence of acetic acid to produce a mixture of ring-brominatedestradiols; isolating 2-bromoestradiol from the mixture of estradiols;and reacting the 2-bromoestradiol with sodium methoxide in the presenceof a copper catalyst to produce 2-methoxyestradiol.
 21. A pharmaceuticalcomposition being substantially free of steroid contaminants havingestrogenic or carcinogenic effects comprising 2-methoxyestradiol havinga purity greater than 98% and containing less than 0.03% estradiol andless than 0.02% estrone produced by the process comprising: protectingthe 3- and 17-hydroxyl groups of estradiol; reacting the protectedestradiol with bromine and acetic acid to produce a 2-brominatedderivative of estradiol; reacting the 2-brominated derivative ofestradiol with sodium methoxide in the presence of a copper catalyst;removing the protecting groups on the 3- and 17-hydroxyl groups toproduce 2-methoxyestradiol; and purifying the 2-methoxyestradiol usingliquid chromatography on an adsorption/partition medium with a solventsystem comprising a polar and a nonpolar solvent.
 22. A pharmaceuticalcomposition being substantially free of steroid contaminants havingestrogenic or carcinogenic effects comprising 2-methoxyestradiol havinga purity greater than 98% and containing less than 0.03% estradiol andless than 0.02% estrone produced by the process comprising:ring-brominating estradiol by reacting estradiol with bromine in thepresence of acetic acid to produce a ring-brominated intermediate;reacting the ring-brominated intermediate with sodium methoxide in thepresence of a copper catalyst to produce 2-methoxyestradiol; andpurifying the 2-methoxyestradiol using liquid chromatography on anadsorption/partition medium with a solvent system comprising a polar anda nonpolar solvent.
 23. A pharmaceutical composition being substantiallyfree of steroid contaminants having estrogenic or carcinogenic effectscomprising 2-methoxyestradiol having a purity greater than 98% andcontaining less than 0.03% estradiol and less than 0.02% estroneproduced by the process comprising: protecting the 3- and 17-hydroxylgroups of estradiol; reacting the protected estradiol with nitric acidand acetic acid to produce a 2-nitro derivative of estradiol; reducingthe 2-nitro derivative of estradiol to produce the corresponding 2-aminoderivative of estradiol; reacting the 2-amino derivative of estradiolunder Sandmeyer conditions to produce a 3-,17-hydroxyl protected2-methoxyestradiol; and removing the protecting groups on the 3- and17-hydroxyl groups to produce 2-methoxyestradiol.
 24. A pharmaceuticalcomposition being substantially free of steroid contaminants havingestrogenic or carcinogenic effects comprising 2-methoxyestradiol havinga purity greater than 98% and containing less than 0.03% estradiol andless than 0.02% estrone produced by the process comprising: protectingthe 3-hydroxyl group of estrone; reacting the protected estrone withnitric acid and acetic acid to produce a 2-nitro derivative of estrone;reducing the 2-nitro derivative of estrone to produce the corresponding2-amino derivative of estrone; reacting the 2-amino derivative ofestrone under Sandmeyer conditions to produce a 3-hydroxyl protected2-methoxyestrone; removing the protecting group on the 3-hydroxyl groupto produce 2-methoxyestrone; and reducing the 17-keto group of2-methoxyestrone to produce 2-methoxyestradiol.
 25. A pharmaceuticalcomposition being substantially free of steroid contaminants havingestrogenic or carcinogenic effects comprising 2-methoxyestradiol havinga purity greater than 98% and containing less than 0.03% estradiol andless than 0.02% estrone produced by the process comprising: brominatingestradiol in the presence of acetic acid to produce a mixture ofring-brominated estradiols; isolating 2-bromoestradiol from the mixtureof estradiols; and reacting the 2-bromoestradiol with sodium methoxidein the presence of a copper catalyst to produce 2-methoxyestradiol.